Most colonial invertebrates have the capacity to discriminate between self tissues and those of unrelated members of the same species. Such allorecognition phenomena have been recognized for over a century, and are often claimed to reflect the evolutionary origins of the vertebrate immune system. Despite the significance of invertebrate allorecognition and its widespread occurrence, the responsible molecules, and the genes encoding them, have not been identified in any invertebrate animal. As a first step to characterize the invertebrate allorecognition determinants, the PI has recently produced a low-resolution genetic map of the chromosomal interval controlling the response in the colonial hydroid Hydractinia. This map delineates a 6.2-centimorgan (cM) region containing at least two linked, highly polymorphic loci. With the map as an essential tool, here it is proposed a molecular and functional characterization of the allorecognition determinants in this lower invertebrate. Specific Aim 1: Generation of a high-resolution linkage map of the Hydractinia allorecognition complex (ARC) at a density of 1 marker per 0.1 cM; Specific Aim 2: Generation of a physical map of the Hydractinia ARC; Specific Aim 3: Identification of candidate allorecognition genes; Specific Aim 4: Functional assays of allorecognition genes.